Our current effort is focusing on the intracellular signals controlling phenomena associated with lymphocyte development differentiation, such as thymic positive and negative selection, lymphocyte activation and generation of immune memory in vivo. Previous analysis indicated that all these phenomena generally require interactions between cell surface antigen receptors and their ligands. It is these interactions that initiate an activation cascade of signaling molecules including tyrosine kinases and G proteins that act concordantly to determine the cellular responses. In the past year, we focused mainly on functional analysis of the c-cbl and Grb2 in using established c-cbl and Grb2 deficient mouse models. Cb1 is the product of the proto-oncogene c-cbl and has been shown to be involved in T cell receptor (TCR) mediated signaling. Thymocytes from c-cbl deficient mice showed hyper-tyrosine phosphorylation on multiple proteins including Zap70 tyrosine kinase, up- regulation of MAP kinase activity and inhibition of phospholipase C-g1(PLC-g1) and PI-3 kinase activities upon stimulation through TCR. While negative selection of the thymocytes remained intact, positive selection of the TCR transgenic T cells was significantly enhanced in these mice. From these results we concluded that Cbl plays a novel role in orchestrating functions of the TCR-mediated multiple signaling pathways and fine-tunes the signaling threshold for thymic selection. We also generated another mouse model deficient in the adaptor molecules Grb2. Analysis indicated that this mutation causes an embryonic lethality at early embryo development. Our preliminary analysis revealed no homozygous Grb2 mutant embryo after day 10 of the pregnancy. This result indicates a critical role of Grb2 molecules for the early embryo-genesis. We are now still in the process of generating a mutant mice with conditional Grb2 deficiency. Finally, in order to study T cells activation in vivo we have generated a mutant mouse strain by introducing a cDNA encoding GFP into the IL-2 gene locus through gene targeting. Our studies indicated that GFP fluorescence is readily detectable upon T cell activation, and is mostly co-expressed with IL-2 at the single- cell level. Upon stimulation through the T cell antigen receptor, antigen-specific CD4+ cells separate into distinct GFP+ and GFP- populations, indicating that activated naive T cells can be divided into IL-2 producers and non-producers. Thus, primary T cells usually express the IL-2 gene biallelically, and are heterogeneous in terms of IL-2 production. Besides, these mice uniquely allow non-invasive detection of IL-2 production by single cells and analysis of the subsequent differentiative fate of these cells as an immune response develops. In the future, we are planning to establish various cell lines from these mutant animals. Detailed analysis of individual signaling pathway will be carried out biochemically using these in vitro systems. Additionally, we will also cross these mutant animals to other animal models in order to understand the contribution of these molecules to immune system physiology and pathology.